Many diseases are thought to be associated with genomic instability. Specific nucleic acid variations, such as mutations, insertions, deletions and other alterations can serve as valuable markers for a variety of diseases, including certain types of cancer. For example, mutations in the BRCA genes have been proposed as indications for breast cancer, and mutations in the p53 cell cycle regulator gene have been associated with numerous cancers, especially colorectal cancer. Other diseases that are linked with genomic instability include, for example, sickle cell anemia, phenylketonuria, hemophilia, and cystic fibrosis. Early detection of these variations allow for early disease diagnosis and provide an avenue for treatment before disease symptoms are presented, when a cure is more readily attainable. It has been suggested that specific variations might be a basis for molecular screening assays for the early stages of certain types of cancer. See, e.g., Sidransky, et al., Science, 256: 102-105 (1992). Therefore, in an effort to detect whether certain variations have occurred and further ascertain whether a person is at risk for developing a disease associated with these variations, molecular screening assays have been developed. However, current screening methods are difficult to manage, lack specificity and are not efficient. Furthermore, such screening assays are not reliable for detecting variations that are present in low frequency in the early stages of diseases such as cancer.
Many cancers are curable if detected early in their development. For example, colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive metastasis, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and black tarry stool or the presence of blood in stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Early detection of colorectal cancer therefore is important in order to significantly reduce its morbidity.
Invasive diagnostic methods, such as endoscopic examination, allow direct visual identification, removal, and biopsy of potentially-cancerous tissue. However, endoscopy is expensive, uncomfortable, inherently risky, and not a practical tool for early diagnosis.
Established non-invasive screening methods involve assaying stool samples for the presence of fecal occult blood or for elevated levels of carcinoembryonic antigen, both of which are suggestive of the presence of colorectal cancer. Additionally, recent developments in molecular biology provide methods of great potential for detecting the presence of a range of DNA variations indicative of colorectal cancer. The presence of such variations can be detected in DNA found in stool samples during various stages of colorectal cancer. However, stool comprises cells and cellular debris from the patient, from microorganisms, and from food, resulting in a heterogeneous population of DNA. This makes detection of small, specific subpopulations difficult to detect reliably.
Attempts have been made to identify and use nucleic acid events that are indicative of cancer and other diseases. However, even when such events are identified, using them to screen patient samples, especially heterogeneous samples, has proven unsuccessful either due to an inability to obtain sufficient sample material, or due to the low sensitivity that results from measuring only a single marker. For example, simply obtaining an adequate amount of human DNA from one type of heterogeneous sample, stool, has proven difficult. See Villa, et al., Gastroenterol., 110: 1346-1353 (1996) (reporting that only 44.7% of all stool specimens, and only 32.6% of stools from healthy individuals produced sufficient DNA for mutation analysis). Other reports in which adequate DNA has been obtained have reported low sensitivity in identifying a patient's disease status based upon a single cancer-associated mutation. See Eguchi, et al., Cancer, 77: 1707-1710 (1996) (using a p53 mutation as a marker for cancer).
Increased knowledge of the molecular basis for disease has lead to a proliferation of screening assays capable of detecting disease-associated nucleic acid variations. One such method identifies a genomic region thought to be associated with a disease and compares the wild-type sequence in that region with the sequence in a patient sample. Differences in the sequences constitute a positive screen. See e.g., Engelke, et al., Proc. Natl. Acad. Sci., 85: 544-548 (1988). However, such methods are time-consuming, costly, and do not have sufficient sensitivity to identify the variation of interest in a heterogeneous sample containing a large amount of non-variant nucleic acid. Thus, sequencing is not practical for large-scale screening assays.
Variations have also been detected by differential hybridization techniques using allele-specific oligonucleotide probes. Saiki et al., Proc. Natl. Acad. Sci., 86: 6230-6234 (1989). Variations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for variation analysis include: (1) the requirement for optimization of hybridization for each probe, (2) the nature of the mismatch and the local sequence impose limitations on the degree of discrimination of the probes, and (3) the difficulty in detecting rare variant nucleic acid molecules in heterogeneous populations of nucleic acid. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is partly due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. In addition, such methods also lack the sensitivity required to detect a small amount of variant nucleic acid in a heterogeneous sample.
A number of detection methods have been developed which are based on template-dependent primer extension. These methods involve hybridizing primers to template nucleic acids and extending the primers using a polymerase. Those methods can be placed into one of two categories: (1) methods using primers which span the region to be interrogated for the variation, and (2) methods using primers which'hybridize upstream of the region to be interrogated for the variation. Typically, the primer extension reaction results in an extended product that can indicate the presence of a variant.
Strategies based on primer extension require considerable optimization to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction. The advantage conferred by the high fidelity of the polymerases can be compromised by the tolerance of nucleotide mismatches in the hybridization of the primer to the template. Any “false” priming will be difficult to distinguish from a true positive signal. The reaction conditions of a primer extension reaction can be optimized to reduce “false” priming due to a mismatched oligonucleotide. However, optimization is labor intensive and expensive, and often results in lower sensitivity due to a reduced yield of extended primer. Also, current primer extension reactions do not reproducibly detect small amounts of variant nucleic acid in biological samples. For this reason, there is a need in the art for additional non-invasive methods for early diagnosis of cancer that will detect early characteristics indicative of the presence of cancer.